Placenta(i99o),
II,
319--3z7
Expression of Human Placenta Alkaline Phosphatase in Placenta During Pregnancy TOMOMITSU O K A M O T O a'b'c, H I S A O S E O a, H I S A O M A N O b, M A D O K A F U R U H A S H I a'b, S E T S U K O G O T O b, YUTAKA TOMODA b & NOBUO MATSUI a ~ Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan b Department of Obstetrics and Gynaecology, School of Medicine, Nagoya University, Nagoya, Japan c To whom all correspondenceshould be addressedat Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, FurooCho, ChikusaKu, Nagoya 464-oi, Japan Paper accepted5.3.1990
SUMMARY To clarify the expression of PLAP during the course of pregnancy, the amount of PLAP mRNA and its activity in normal placental villi were measured. Both PLAP and its mRNA were found in placentae of as early as 7 weeks of gestation, and they continued to increase throughout pregnancy. But they showed different patterns of increase. The amount of PLAP mRNA began to increase dramatically around I3th week and probably continued to increase gradually until term. PLAP activity per gram of villi showed a gradual increase from around I3th week and a marked increase was observed after about 2oth week. PLAP levels in sera from pregnant women were also measured, and they showed a pattern of increase imilar to that of PLAP activity per gram of villi. The continuous increase in the expression of PLAP throughout pregnancy suggests that PLAP may play a role in feto-maternal metabolism and placental differentiation. INTRODUCTION Human placental alkaline phosphatase (PLAP) is a glycoprotein synthesized in syncytrophoblasts and located mainly on the plasma membrane (Hulstaert et al, i973; Carlson, Wada and Sussman, I976 ). The enzyme is a dimer composed of identical subunits of approximately 65 kDa (Holmgren and Stigbrand, 1976). PLAP is released into the maternal circulation and its plasma concentration increases as pregnancy proceeds (Ronin-Walknowska et al, I984; Contractor et al, I985). oi43-4oo4/9o/o4o3 I9 + 09 $o5.oo/o
9 199o Bailli~re Tindall Ltd
3zo
Placenta 0990), Vol. t z
There are at least three isoenzymes in human alkaline phosphatase, i.e. tissue unspecific type (present in liver, bone and kidney), intestinal type, and placental type. This enzyme is mainly localized where transport process is important, e.g. on the microvillar membrane of the proximal tubular cells in the kidney or of the epithelial cells in the intestinal mucosa. In fact, the intestinal type has been suggested to be involved in fat absorption (Glickman et al, x97o; Linscheer, Malagelada and Fishman, 1970, or in vitamin D-mediated calcium transport (Norman et al, x97o ). Since placental villi are located where maternal blood has direct access to the fetus, PLAP could play an important role in the transport of nutrients from mother to fetus. Clinically, PLAP has been used as an index of placental function or fetal well-being; low PLAP values in maternal serum were found in pregnancies with intrauterine growth retardation (Holmgren et al, I979; Ronin-Walknowska et al, I984). T h e elevated levels from early stage of pregnancy were observed under ABO and Rh incompatible status (Lucarelli et al, I985). PLAP is also of special interest because it is ectopically expressed in some malignant tumours, and it has been used as a tumour marker for ovarian cancer (McDicken et al, 1985), seminoma (Tucker et al, I 9 8 5 ) , o r intracranial germinoma (Shinoda et al, 1988 ), Although PLAP expression is induced by agents such as cAMP or sodium butyrate in cancer cell lines (Hamilton and Sussman, 198I ; Ito and Chou, I984), little is known about the developmental change in the expression of PLAP in normal placenta during pregnancy. Here we studied the expression of PLAP in the normal human placenta during pregnancy. We also investigated changes of PLAP levels in sera of pregnant women.
MATERIALS AND METHODS Tissue samples Placental tissues were obtained from 23 normal subjects of the t s t (7--I I weeks of gestation, n = io), the 2nd (i3-23 weeks, n -- 6), and the 3rd (31-39 weeks, n = 7) trimester. First trimester placentae were obtained at the time of therapeutic abortions. Second trimester placentae and a placenta at the 31st week were collected at premature deliveries and 3rd trimester placentae at term deliveries. Villous portions were excised and washed extensively in phosphate-buffered saline, frozen immediately in liquid nitrogen, and stored at - 8 o ~ until use. The same samples were used for the measurement of PLAP m R N A and PLAP activities. Similarly, normal liver tissue attached to hepatocellular carcinoma was obtained at surgery, washed and stored. Blood s a m p l e s Serum samples were obtained from 242 women with normal pregnant course (8-40 weeks of gestation), and stored at - 8o~ until use. R N A isolation RNA was isolated by the guanidinium-thiocyanate method of Chomczynski and Sacchi (I987). Frozen pieces of tissue (1 g) were pulverized in liquid nitrogen and homogenized in 7 ml of Solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, p H 7.o, o.5 per cent sarcosyl, o.r M z-mercaptoethanol). Sequentially, 0.5 ml of 2 M sodium acetate, p H 4.o, 5 ml of phenol (water-saturated), and i ml of chloroform-isoamylalcohol mixture (49:1) were added to the homogenate and cooled on ice for I5 min. T h e samples were centrifuged at Io ooo g for 20 min at 4~ the aqueous phase was harvested, mixed with equal volume of isopropanol and
Okamoto et al: Placental Alkaline Phosphatase
32i
placed at - 3o~ for at least 1 h. After centrifugation at IO ooo g for 2o min at 4~ the pellet was dissolved in o.7 ml of Solution D and precipitated with o.7 ml of isopropanol at - 3 o ~ for i h. RNA was recovered by centrifugation at io ooo g for 2o min at 4~ washed in 7 ~ per cent ethanol, vacuum dried, dissolved in distilled water and stored at --8o~ until analysis. The amount of RNA was determined by measuring absorbance at 26o nm. Poly(A) + R N A was prepared by oligo(dT) cellulose chromatography (Aviv and Leder, i972 ). P r o b e a n d its labelling with 32p The probe for PLAP m R N A is a kind gift from Dr J. L. Millfin (La Jolla Cancer Research Foundation, La Jolla, California). The 2.8-kilobase (kb) c D N A includes approximately 1.7 kb of coding region and i.i kb of 3' untranslated sequence (Millfin, 1986 ). The c D N A was labelled by deoxycytidine 5'-[g-32p]triphosphate ([32p]dCTP) using the method of randomprimed D N A labelling described by Feinberg and Vogelstein (i983). N o r t h e r n blot analysis Total RNA (I8.5#g) was denatured by heating at 5o~ for 15min in the presence of I M glyoxal and 5o per cent dimethylsulphoxide, separated by electrophoresis on o.8 per cent agarose gel, and transferred to nylon membranes (Gene Screen Plus, New England Nuclear, Boston, Massachusetts) in IO x standard saline citrate (SSC) (I x SSC = o.15 M sodium chloride/o.ol5 M sodium citrate, pH 7.o). After the transfer, the blots were briefly placed in a 5o mM NaOH solution and then in a solution of i x S S C - 0.2 M Tris-HCl (pH 7.5) to reverse glyoxal reaction, and air dried. Prehybridization was carried out for 3 h at 42~ in 5o per cent (v/v) formamide containing 5 x SSC, 5 x SSPE (i x SSPE = o.15 M N a C l - I o mM N a O H 2 P O 4 H 2 0 - I mM EDTA), i per cent sodium dodecyl sulphate (SDS), 1oo/zg/ml denatured herring sperm DNA, and o. i per cent (w/v) each of BSA, Ficoll and polyvinylpyrrolidone. Hybridization was carried out for 16 h at 42~ in the same buffer used in prehybridization, to which 32p-labelled PLAP c D N A insert (lO 6 cpm/ml) was added. The blots were washed twice in 2 x SSC at room temperature for 5 min, twice in 2 x SSC-1 per cent SDS at 65~ for 3o min, and then twice in o.i x SSC for 3o min at room temperature. After washing, the blots were exposed to X-ray film ( X - O M A T AR, Eastman Kodak, Rochester, New York) and autoradiographed for 2-6 days at - 8o~ D o t blot analysis Total R N A was denatured by heating at 6o~ for 15 min in 6 x SSC containing 2o per cent (v/v) formaldehyde. Serial twofold dilutions of the denatured R N A (containing 2 ~ 2 . 5 #g) were blotted to Gene Screen Plus hybridization membrane according to Thomas (Thomas, 1983), baked at 8o~ for 2 h, and hybridized as described in Northern blot analysis. Autoradiography was performed for 3o h at - 8 o ~ The amount of P L A P m R N A was measured by scanning densitometry of the autoradiograms. P L A P e x t r a c t i o n f r o m the tissue PLAP was extracted from the tissue according to the method of Sakiyama, Robinson and Chou (I978). Approximately o.5 g of villous tissue was homogenized in 3 ml of iomM Tris-HCl, pH 7.4, and sonicated for 2 min. n-Butanol was added to the homogenate (final concentration 3o per cent, v/v), and the mixture was stirred at 4~ for i6 h. After centrifugation at IO ooog for 15 rain, the aqueous phase was harvested and dialyzed against I0mM Tris-HCl, pH7.4, containing i mM MgCI 2 at 4~ for 16 h. One hundred microliter aliquots or diluted samples were used for the determination of PLAP.
3~
Placenta
099o),
Vol. t z
Measurement of PLAP activity P L A P activity in serum or tissue was measured by the enzyme immunocatalytic assay using a monoclonal antibody against P L A P (Kinoshita et al, in press). Flat-bottom, 96-well microtiter plates (Nunc-Immunoplate I; Nunc, Denmark) were coated with ioo #1 of 2 mg/l solution of the purified monoclonal antibody in o.I M carbonate-bicarbonate buffer (pH9.6), and incubated at 4~ for i6 h. T h e antibody solution was aspirated off, the plate was washed once with the washing buffer (IOmM Tris-HCl, p H 7-4, containing o.o5 per cent Tween 2o), and the wells were filled with zoo/tl of i o mM Tris-HCl, p H 7.4, containing o. 15 M NaCI, 2 per cent BSA and o.i per cent sodium azide. After incubation at 4~ for i 6 h , IOo/Ul of the sample diluted optimally in i o mM T r i s - H C l (pH 7.4), containing o.3 M NaCI, o. I per cent BSA, I mM MgCI2, 0.5 per cent gelatin (Difco Labs; Detroit, Michigan) and o. I per cent sodium azide was added, incubated for 2 h at room temperature, and washed four times with the washing buffer. Then I5o/al of i mg/ml of p-nitrophenylphosphate (Sigma; St Louis, Missouri) in o.I M diethanolamine-HCl buffer (pH 9.8) was added to each well, followed by incubation at 37~ for 2 h. T h e absorbance of the solution in each well was read at 405 nm by EASY R E A D E R EAR 4oo ( S L T - L A B I N S T R U M E N T S , Salzburg, Austria). T h e minimum detectable concentration in this assay was i.o • lO -3 m l U per well (defined as a concentration difference from o at the 99.9 per cent confidence). Both the intra- and inter-assay coefficients of variation were within io per cent. T h e monoclonal antibody showed no cross-reaction with the tissue unspecific alkaline phosphatase up to the concentration of2.o • lO 2 m l U per well.
RESULTS Figure I shows Northern blot analysis o f P L A P m R N A in the placentae from the ist, and and
Gestational week
7
8
9
13
17
23
38
39
39
2 . 9 k b ,-=,)p
Figure t. Northern blot analysis of PLAP mRNA. Total RNA (I8.5,ttg) was denatured by glyoxaland separated on 0.8 per cent agarosegels, transferred to a nylon membrane (Gene Screen Plus) and hybridized with 32p-labelledPLAP cDNA insert. Autoradiography was carried out for 2 days (13-39 weeks) or for 6 days (7~) weeks). Three samples from each trimester were analyzed. The size of PLAP mRNA was estimated to be 2.9 kb from the migration of molecularweight marker in the same run. The size marker used was bacteriophage2 DNA digested with Hind III and EcoRI obtained from Wako Pure Chemical Industries, Osaka,Japan.
Okamoto et ah Placental Alkafine Phosphatase
3a3
q
q
q
q
2.9 kb --)
total RNA
poly(A)+RNA
Figure 2. Northern blot analysis of RNA from liver and the placenta. Two human liver RNAs and two placental RNAs (i 7 and 39 weeks of gestation) were analysed. Total RNA (I 8.5 #g; left column) or poly(A) + RNA (5 ~tg; right column) was blotted and hybridized with FLAP cDNA. No cross-hybridization was observed between PLAN cDNA and liver RNA.
3rd trimester. A single FLAP mRNA with a size of 2. 9 kb was demonstrated and the amount of FLAP mRNA increased as pregnancy progressed. Northern blot analysis of both total and poly(A) + RNA from liver revealed no cross-hybridization of FLAP probe with mRNA coding tissue unspecific alkaline phosphatase (Figure 2). The increase of FLAP mRNA during pregnancy was again demonstrated.
r =0.86
I00
9
.~
~8o
(P
II,
319--3z7
Expression of Human Placenta Alkaline Phosphatase in Placenta During Pregnancy TOMOMITSU O K A M O T O a'b'c, H I S A O S E O a, H I S A O M A N O b, M A D O K A F U R U H A S H I a'b, S E T S U K O G O T O b, YUTAKA TOMODA b & NOBUO MATSUI a ~ Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan b Department of Obstetrics and Gynaecology, School of Medicine, Nagoya University, Nagoya, Japan c To whom all correspondenceshould be addressedat Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, FurooCho, ChikusaKu, Nagoya 464-oi, Japan Paper accepted5.3.1990
SUMMARY To clarify the expression of PLAP during the course of pregnancy, the amount of PLAP mRNA and its activity in normal placental villi were measured. Both PLAP and its mRNA were found in placentae of as early as 7 weeks of gestation, and they continued to increase throughout pregnancy. But they showed different patterns of increase. The amount of PLAP mRNA began to increase dramatically around I3th week and probably continued to increase gradually until term. PLAP activity per gram of villi showed a gradual increase from around I3th week and a marked increase was observed after about 2oth week. PLAP levels in sera from pregnant women were also measured, and they showed a pattern of increase imilar to that of PLAP activity per gram of villi. The continuous increase in the expression of PLAP throughout pregnancy suggests that PLAP may play a role in feto-maternal metabolism and placental differentiation. INTRODUCTION Human placental alkaline phosphatase (PLAP) is a glycoprotein synthesized in syncytrophoblasts and located mainly on the plasma membrane (Hulstaert et al, i973; Carlson, Wada and Sussman, I976 ). The enzyme is a dimer composed of identical subunits of approximately 65 kDa (Holmgren and Stigbrand, 1976). PLAP is released into the maternal circulation and its plasma concentration increases as pregnancy proceeds (Ronin-Walknowska et al, I984; Contractor et al, I985). oi43-4oo4/9o/o4o3 I9 + 09 $o5.oo/o
9 199o Bailli~re Tindall Ltd
3zo
Placenta 0990), Vol. t z
There are at least three isoenzymes in human alkaline phosphatase, i.e. tissue unspecific type (present in liver, bone and kidney), intestinal type, and placental type. This enzyme is mainly localized where transport process is important, e.g. on the microvillar membrane of the proximal tubular cells in the kidney or of the epithelial cells in the intestinal mucosa. In fact, the intestinal type has been suggested to be involved in fat absorption (Glickman et al, x97o; Linscheer, Malagelada and Fishman, 1970, or in vitamin D-mediated calcium transport (Norman et al, x97o ). Since placental villi are located where maternal blood has direct access to the fetus, PLAP could play an important role in the transport of nutrients from mother to fetus. Clinically, PLAP has been used as an index of placental function or fetal well-being; low PLAP values in maternal serum were found in pregnancies with intrauterine growth retardation (Holmgren et al, I979; Ronin-Walknowska et al, I984). T h e elevated levels from early stage of pregnancy were observed under ABO and Rh incompatible status (Lucarelli et al, I985). PLAP is also of special interest because it is ectopically expressed in some malignant tumours, and it has been used as a tumour marker for ovarian cancer (McDicken et al, 1985), seminoma (Tucker et al, I 9 8 5 ) , o r intracranial germinoma (Shinoda et al, 1988 ), Although PLAP expression is induced by agents such as cAMP or sodium butyrate in cancer cell lines (Hamilton and Sussman, 198I ; Ito and Chou, I984), little is known about the developmental change in the expression of PLAP in normal placenta during pregnancy. Here we studied the expression of PLAP in the normal human placenta during pregnancy. We also investigated changes of PLAP levels in sera of pregnant women.
MATERIALS AND METHODS Tissue samples Placental tissues were obtained from 23 normal subjects of the t s t (7--I I weeks of gestation, n = io), the 2nd (i3-23 weeks, n -- 6), and the 3rd (31-39 weeks, n = 7) trimester. First trimester placentae were obtained at the time of therapeutic abortions. Second trimester placentae and a placenta at the 31st week were collected at premature deliveries and 3rd trimester placentae at term deliveries. Villous portions were excised and washed extensively in phosphate-buffered saline, frozen immediately in liquid nitrogen, and stored at - 8 o ~ until use. The same samples were used for the measurement of PLAP m R N A and PLAP activities. Similarly, normal liver tissue attached to hepatocellular carcinoma was obtained at surgery, washed and stored. Blood s a m p l e s Serum samples were obtained from 242 women with normal pregnant course (8-40 weeks of gestation), and stored at - 8o~ until use. R N A isolation RNA was isolated by the guanidinium-thiocyanate method of Chomczynski and Sacchi (I987). Frozen pieces of tissue (1 g) were pulverized in liquid nitrogen and homogenized in 7 ml of Solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, p H 7.o, o.5 per cent sarcosyl, o.r M z-mercaptoethanol). Sequentially, 0.5 ml of 2 M sodium acetate, p H 4.o, 5 ml of phenol (water-saturated), and i ml of chloroform-isoamylalcohol mixture (49:1) were added to the homogenate and cooled on ice for I5 min. T h e samples were centrifuged at Io ooo g for 20 min at 4~ the aqueous phase was harvested, mixed with equal volume of isopropanol and
Okamoto et al: Placental Alkaline Phosphatase
32i
placed at - 3o~ for at least 1 h. After centrifugation at IO ooo g for 2o min at 4~ the pellet was dissolved in o.7 ml of Solution D and precipitated with o.7 ml of isopropanol at - 3 o ~ for i h. RNA was recovered by centrifugation at io ooo g for 2o min at 4~ washed in 7 ~ per cent ethanol, vacuum dried, dissolved in distilled water and stored at --8o~ until analysis. The amount of RNA was determined by measuring absorbance at 26o nm. Poly(A) + R N A was prepared by oligo(dT) cellulose chromatography (Aviv and Leder, i972 ). P r o b e a n d its labelling with 32p The probe for PLAP m R N A is a kind gift from Dr J. L. Millfin (La Jolla Cancer Research Foundation, La Jolla, California). The 2.8-kilobase (kb) c D N A includes approximately 1.7 kb of coding region and i.i kb of 3' untranslated sequence (Millfin, 1986 ). The c D N A was labelled by deoxycytidine 5'-[g-32p]triphosphate ([32p]dCTP) using the method of randomprimed D N A labelling described by Feinberg and Vogelstein (i983). N o r t h e r n blot analysis Total RNA (I8.5#g) was denatured by heating at 5o~ for 15min in the presence of I M glyoxal and 5o per cent dimethylsulphoxide, separated by electrophoresis on o.8 per cent agarose gel, and transferred to nylon membranes (Gene Screen Plus, New England Nuclear, Boston, Massachusetts) in IO x standard saline citrate (SSC) (I x SSC = o.15 M sodium chloride/o.ol5 M sodium citrate, pH 7.o). After the transfer, the blots were briefly placed in a 5o mM NaOH solution and then in a solution of i x S S C - 0.2 M Tris-HCl (pH 7.5) to reverse glyoxal reaction, and air dried. Prehybridization was carried out for 3 h at 42~ in 5o per cent (v/v) formamide containing 5 x SSC, 5 x SSPE (i x SSPE = o.15 M N a C l - I o mM N a O H 2 P O 4 H 2 0 - I mM EDTA), i per cent sodium dodecyl sulphate (SDS), 1oo/zg/ml denatured herring sperm DNA, and o. i per cent (w/v) each of BSA, Ficoll and polyvinylpyrrolidone. Hybridization was carried out for 16 h at 42~ in the same buffer used in prehybridization, to which 32p-labelled PLAP c D N A insert (lO 6 cpm/ml) was added. The blots were washed twice in 2 x SSC at room temperature for 5 min, twice in 2 x SSC-1 per cent SDS at 65~ for 3o min, and then twice in o.i x SSC for 3o min at room temperature. After washing, the blots were exposed to X-ray film ( X - O M A T AR, Eastman Kodak, Rochester, New York) and autoradiographed for 2-6 days at - 8o~ D o t blot analysis Total R N A was denatured by heating at 6o~ for 15 min in 6 x SSC containing 2o per cent (v/v) formaldehyde. Serial twofold dilutions of the denatured R N A (containing 2 ~ 2 . 5 #g) were blotted to Gene Screen Plus hybridization membrane according to Thomas (Thomas, 1983), baked at 8o~ for 2 h, and hybridized as described in Northern blot analysis. Autoradiography was performed for 3o h at - 8 o ~ The amount of P L A P m R N A was measured by scanning densitometry of the autoradiograms. P L A P e x t r a c t i o n f r o m the tissue PLAP was extracted from the tissue according to the method of Sakiyama, Robinson and Chou (I978). Approximately o.5 g of villous tissue was homogenized in 3 ml of iomM Tris-HCl, pH 7.4, and sonicated for 2 min. n-Butanol was added to the homogenate (final concentration 3o per cent, v/v), and the mixture was stirred at 4~ for i6 h. After centrifugation at IO ooog for 15 rain, the aqueous phase was harvested and dialyzed against I0mM Tris-HCl, pH7.4, containing i mM MgCI 2 at 4~ for 16 h. One hundred microliter aliquots or diluted samples were used for the determination of PLAP.
3~
Placenta
099o),
Vol. t z
Measurement of PLAP activity P L A P activity in serum or tissue was measured by the enzyme immunocatalytic assay using a monoclonal antibody against P L A P (Kinoshita et al, in press). Flat-bottom, 96-well microtiter plates (Nunc-Immunoplate I; Nunc, Denmark) were coated with ioo #1 of 2 mg/l solution of the purified monoclonal antibody in o.I M carbonate-bicarbonate buffer (pH9.6), and incubated at 4~ for i6 h. T h e antibody solution was aspirated off, the plate was washed once with the washing buffer (IOmM Tris-HCl, p H 7-4, containing o.o5 per cent Tween 2o), and the wells were filled with zoo/tl of i o mM Tris-HCl, p H 7.4, containing o. 15 M NaCI, 2 per cent BSA and o.i per cent sodium azide. After incubation at 4~ for i 6 h , IOo/Ul of the sample diluted optimally in i o mM T r i s - H C l (pH 7.4), containing o.3 M NaCI, o. I per cent BSA, I mM MgCI2, 0.5 per cent gelatin (Difco Labs; Detroit, Michigan) and o. I per cent sodium azide was added, incubated for 2 h at room temperature, and washed four times with the washing buffer. Then I5o/al of i mg/ml of p-nitrophenylphosphate (Sigma; St Louis, Missouri) in o.I M diethanolamine-HCl buffer (pH 9.8) was added to each well, followed by incubation at 37~ for 2 h. T h e absorbance of the solution in each well was read at 405 nm by EASY R E A D E R EAR 4oo ( S L T - L A B I N S T R U M E N T S , Salzburg, Austria). T h e minimum detectable concentration in this assay was i.o • lO -3 m l U per well (defined as a concentration difference from o at the 99.9 per cent confidence). Both the intra- and inter-assay coefficients of variation were within io per cent. T h e monoclonal antibody showed no cross-reaction with the tissue unspecific alkaline phosphatase up to the concentration of2.o • lO 2 m l U per well.
RESULTS Figure I shows Northern blot analysis o f P L A P m R N A in the placentae from the ist, and and
Gestational week
7
8
9
13
17
23
38
39
39
2 . 9 k b ,-=,)p
Figure t. Northern blot analysis of PLAP mRNA. Total RNA (I8.5,ttg) was denatured by glyoxaland separated on 0.8 per cent agarosegels, transferred to a nylon membrane (Gene Screen Plus) and hybridized with 32p-labelledPLAP cDNA insert. Autoradiography was carried out for 2 days (13-39 weeks) or for 6 days (7~) weeks). Three samples from each trimester were analyzed. The size of PLAP mRNA was estimated to be 2.9 kb from the migration of molecularweight marker in the same run. The size marker used was bacteriophage2 DNA digested with Hind III and EcoRI obtained from Wako Pure Chemical Industries, Osaka,Japan.
Okamoto et ah Placental Alkafine Phosphatase
3a3
q
q
q
q
2.9 kb --)
total RNA
poly(A)+RNA
Figure 2. Northern blot analysis of RNA from liver and the placenta. Two human liver RNAs and two placental RNAs (i 7 and 39 weeks of gestation) were analysed. Total RNA (I 8.5 #g; left column) or poly(A) + RNA (5 ~tg; right column) was blotted and hybridized with FLAP cDNA. No cross-hybridization was observed between PLAN cDNA and liver RNA.
3rd trimester. A single FLAP mRNA with a size of 2. 9 kb was demonstrated and the amount of FLAP mRNA increased as pregnancy progressed. Northern blot analysis of both total and poly(A) + RNA from liver revealed no cross-hybridization of FLAP probe with mRNA coding tissue unspecific alkaline phosphatase (Figure 2). The increase of FLAP mRNA during pregnancy was again demonstrated.
r =0.86
I00
9
.~
~8o
(P
